This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protective effects of estradiol (i.e., estrogen) against coronary artery disease (CAD) in women may be due to the action of its two primary metabolites, 2-hydroxyestradiol and 2-methoxyestradiol. These experiments will be conducted on the smooth muscle of coronary arteries from two different age groups (9 months old versus 4 years old) of sexually mature female Yorkshire pigs. The hypothesis of this project is that 2-methoxyestradiol is more efficacious than 2-hydroxyestradiol in protecting against CAD by reducing arterial tone and decreasing the proliferation of smooth muscle cells. The specific aims are to investigate the impact of aging, and 2-hydroxyestradiol and 2-methoxyestradiol on (1) the intracellular Ca2+-induced smooth muscle contraction, (2) the Ca2+-activated big potassium (BKCa) channel mediated arterial relaxation, and (3) smooth muscle cell proliferation. The methods for Aim #1 are to use fluorescent microscopy to measure intracellular Ca2+ concentration ([Ca]i) changes in response to agonist-induced (K+ and endothelin-1, respectively) Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum upon incubation of the smooth muscle cells in 2-hydroxyestradiol and 2-methoxyestradiol. Then it will be determined if the [Ca]i changes produce changes in isometric contraction of arterial rings. For Aim #2, whole cell patch clamp techniques will be used to measure BKCa channel current after exposing the isolated smooth muscle cells to 2-hydroxyestradiol or 2-methoxyestradiol. Next, the isometric relaxation of arterial rings will be investigated in response to 2-hydroxyestradiol or 2-methoxyestradiol in the presence of selective BKCa channel antagonists. For Aim #3, bromodeoxyuridine (BrdU) incorporation during the cell cycle will be used as a measure of DNA synthesis. A fluorescently tagged antibody to BrdU and FM will be used to measure BrdU incorporation into DNA. The overall relevance of this project is that the metabolites of estrogen may protect against CAD without causing estrogen-induced tumor growth.